[[alternative]]Purification and characterization of a chitinase from pseudomonas sp.TKU008

碩士[[abstract]]本研究以蝦蟹殼粉（SCSP）為 Pseudomonas sp.TKU008 生產蛋白酶與幾丁質酶之唯一碳/氮源，於含有 1% SCSP、0.05% MgSO4．7H2O、0.1% K2HPO4（pH7）之液態培養基搖瓶培養 4 天可得較高酵素活性，將所得發酵上清液進行酵素之純化分離及定性。首先將所得發酵上清液經硫酸銨沉澱濃縮、透析去除鹽類，再經過 DEAE-Sepharose、Phenyl Sepharose 及 Sephacryl S-100 管柱層析等分離步驟，純化出一種幾丁質酶，經 SDS-PAGE 測其分子量為 40 kDa。純化後幾丁質酶之活性回收率為 9%，純化倍數為 24.1倍，比活性為 0.175 U/mg。以懸浮態幾丁質為基質測得幾丁質酶最適pH值、最適反應溫度、pH安定性及熱安定性分別為 pH7、50℃、pH6～8 及 <50℃；幾丁質酶活性受 Cu2+、Mn2+所抑制。 由幾丁質酶的液相層析串連式質譜分析得到胺基酸序列片段，序列比對有 26％ 序列接近 Bacillus cereus 的 chitinase B（GenBank accession number gi 10944304）。[[abstract]]The protease and chitinase producing strain, Pseudomonas sp.TKU008, was isolated from the soil in Taiwan. The shrimp and crab shell powder was used as sole carbon / nitrogen source. The optimized condition for protease and chitinase production were found when the culture was shaken at 30℃ for 4 days in 100mL of medium contain 1% shrimp and crab shell powder （SCSP）, 0.1% K2HPO4 and 0.05% MgSO4．7H2O （pH7）.One chitinase was purified by chromatography procedures of DEAE-sepharose CL-6B, Phenyl Sepharose and Sephacryl S-100. The molecular weight of the chitinase was 40k Da estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitinase were pH7, 50°C, pH6～8, and